Authors

Document Type

Article

Disciplines

Biochemistry

Abstract

The determination of urinary 3-methoxy-4-hydroxyphenylethylene glycol (HMPG), a major metabolite of epinephrine and norepinephrine by gas-liquid chromatography and electron capture detection is described. A purified extract of HMPG was treated with trifluoroacetic anhydride to form the highly sensitive HMPG-trifluoroacetate derivative. Chromatography was performed on a 6% QF-I- 0065 column at 158°. Recovery was found to be 55·4% using [3H]HMPG. Two sets of eight identical samples demonstrated a standard deviation from the mean of 6.9% and 5.7%. Thirty-five normal human urine specimens assayed by this procedure gave a mean HMPG excretion of 0.86 #gfmg creatinine (range 0.25-1.49). Six urine samples from patients with proven pheochromocytoma, and four from patients with proven neuroblastoma were analyzed and gave rise to the anticipated elevation in HMPG excretion. The high sensitivity of this procedure points to the possibility of estimation of plasma HMPG levels. The isolation and characterization of 3-methoxy-4-hydroxyphenylethylene glycol (HMPG) as a metabolite of epinephrine and norepinephrine was reported by Axelrod et al. in 1959. This compound normally present in urine as a sulfate conjugate is presumably formed by the reduction of the unstable intermediate 3-methoxy- 4-hyd.roxymandelic aldehyde, the aldehyde also being the common precursor via oxidation of 3-methoxy-4-hydroxymandelic acid (VMA). Early attempts by Gitlow et al. to quantitate the normal urinary levels of HMPG utilized the two-dimensional chromatographic system of Armstrong. Values of approximately I μg/mg creatinine were found. The accuracy of this method was limited by the unfavorable RF of HMPG in this system, giving rise to a rather diffuse spot. The only published procedure for determination of HMPG is that of Ruthven and Sandler. HMPG is determined spectrophotometrically after conversion to vanillin. The values reported are higher than those obtained by paper chromatography, indicating the presence of other substances absorbing at 360 mμ. The combination of gas-liquid chromatography (GLC) and electron capture detection offers a powerful means for the analysis of trace amounts of substances present in a complex mixture. Previous studies on the electron capture properties of trifiuoroacetates and the GLC assay of VMA as 0-trifluoroacetyl vanillin indicated the usefulness of trifiuoroacetylation as the reaction of choice for converting HMPG to a derivative possessing excellent electron capture as well as chromatographic properties